Application of substance for increasing content or activity of OSKM in prevention of articular cartilage senescence and treatment of osteoarthritis disease

Abstract

The invention discloses an application of a substance for increasing the content or activity of OSKM in preventing articular cartilage senescence and treating osteoarthritis diseases. The OSKM provided by the invention is obtained by connecting Oct4, Sox2, Klf4 and C-Myc. Experiments prove that OSKM or a substance for increasing the content or activity of OSKM has a definite effect in preventing cartilago articularis senescence and treating osteoarthritis diseases, and a new strategy is provided for developing gene therapy for preventing cartilago articularis senescence and treating osteoarthritis diseases. And the selectable range of clinical gene therapy and drug therapy is expanded.

Description

Technical Field

[0001] The present invention relates to the application of a substance for increasing the content or activity of OSKM in the field of biomedicine in preventing the aging of articular cartilage and treating osteoarthritis. Background Art

[0002] Osteoarthritis is a chronic joint disease that is more common in elderly individuals. It is caused by cartilage wear and degeneration, which leads to increased inflammation, compensatory osteophyte hyperplasia and synovial hyperplasia, and is usually accompanied by joint pain and deformation. For the treatment of osteoarthritis, non-steroidal anti-inflammatory drugs and weight loss are usually taken to relieve joint pain to a certain extent, but the effect of long-term cure is limited. There is a group of cells with self-renewal ability (cartilage stem / progenitor cells) on the surface of cartilage. When cartilage tissue is damaged, cartilage stem / progenitor cells will differentiate into chondrocytes for repair. In the process of osteoarthritis, cartilage stem / progenitor cells age, causing the regeneration and repair speed of cartilage to be lower than the degeneration speed, resulting in cartilage thinning and lesions, inducing arthritis. It has been reported that gene therapy can be used to introduce factors such as DGCR8, CBX4, YAP1, FOXD1 and CLOCK into the joint cavity, which can slow down the aging of cartilage stem / progenitor cells, promote cartilage regeneration, and restore the physiological function of joints.

[0003] The four reprogramming factors OSKM (Oct4, Sox2, Klf4, C-Myc) can reprogram fibroblasts into induced pluripotent stem cells (iPS cells) in vitro. Intermittent expression of OSKM can alleviate the aging of tissues such as liver, muscle, kidney, and retina, and prolong the life span of mice. So far, no studies have shown the role of OSKM in the treatment or relief of osteoarthritis. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to treat and / or prevent osteoarthritis, or delay joint aging.

[0005] In order to solve the above technical problems, the present invention first provides the use of a substance that reprograms four-factor OSKM or increases the content or activity of OSKM in the preparation of a product for treating and / or preventing osteoarthritis.

[0006] In the above applications, OSKM may be composed of Oct4, Sox2, Klf4 and C-Myc or may be linked to Oct4, Sox2, Klf4 and C-Myc.

[0007] In one embodiment of the present invention, OSKM is linked sequentially by Oct4, Sox2, Klf4 and C-Myc.

[0008] Oct4 may be the protein shown in positions 1-352 of SEQ ID No. 2 or positions 1-360 of SEQ ID No. 5;

[0009] Sox2 may be the protein shown at positions 379-697 of SEQ ID No. 2 or at positions 387-703 of SEQ ID No. 5;

[0010] Klf4 may be the protein shown at positions 732-1205 of SEQ ID No. 2 or positions 738-1207 of SEQ ID No. 5;

[0011] C-Myc may be the protein shown at positions 1249-1687 of SEQ ID No.2 or at positions 1251-1689 of SEQ ID No.5.

[0012] In SEQ ID No. 5, positions 1-360 represent OCT4, positions 387-703 represent SOX2, positions 738-1207 represent KLF4, and positions 1251-1689 represent C-MYC.

[0013] Specifically, OSKM may be the following A1), A2) or A3):

[0014] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0015] A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0016] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0017] In order to facilitate the purification of OSKM in A1), a tag as shown in the following table may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0018] Table: Sequence of tags

[0019] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL

[0020] The OSKM protein in A2) is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID No.2 and having the same function. The 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. Specifically, the OSKM protein in A2) can be the protein shown in SEQ ID No.5.

[0021] The OSKM protein in A2) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0022] The gene encoding the OSKM protein in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No.1 or SEQ ID No.4, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in the above table to its 5' end and / or 3' end. Among them, the DNA molecule shown in SEQ ID No.1 encodes the OSKM protein shown in SEQ ID No.2, and the DNA molecule shown in SEQ ID No.4 encodes the OSKM protein shown in SEQ ID No.5.

[0023] In SEQ ID No. 4, positions 1-1080 represent OCT4, positions 1081-1158 represent P2A, positions 1159-2109 represent SOX2, positions 2110-2211 represent T2A, positions 2212-3621 represent KLF4, positions 3622-3750 represent E2A, and positions 3751-5070 represent C-MYC.

[0024] In the above application, the substance that increases the content or activity of OSKM can be a substance that promotes the expression of OSKM gene or a biological material related to OSKM;

[0025] The biological material is any one of the following B1) to B4):

[0026] B1) a nucleic acid molecule encoding OSKM;

[0027] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0029] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0030] In the above application, the nucleic acid molecule described in B1) may be the following b11) or b12) or b13) or b14):

[0031] b11) the coding sequence is the DNA molecule of SEQ ID No.1 in the sequence list;

[0032] b12) the DNA molecule shown in SEQ ID No. 1 in the sequence list;

[0033] b13) a DNA molecule encoding OSKM that has 75% or more identity with the nucleotide sequence defined in b11) or b12);

[0034] b14) A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b11) or b12) or b13) and encodes OSKM.

[0035] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0036] Those skilled in the art can easily mutate the nucleotide sequence encoding the OSKM protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the OSKM protein isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the OSKM protein and have the function of the OSKM protein.

[0037] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the present invention encoding the protein composed of the amino acid sequence shown in SEQ ID No.2. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Specifically, the DNA molecule described in b13) can be as shown in SEQ ID No.4.

[0038] In the above application, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 2×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 0.5×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 0.1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 0.1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M The membrane can be hybridized in a mixed solution of NaPO4 and 1mM EDTA, and rinsed in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, the membrane can be hybridized in a solution of 6×SSC, 0.5% SDS at 65°C, and then washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS. Alternatively, the membrane can be hybridized in a solution of 2×SSC, 0.1% SDS at 68°C and washed twice for 5 minutes each time, and then hybridized in a solution of 0.5×SSC, 0.1% SDS at 68°C and washed twice for 15 minutes each time. Alternatively, the membrane can be hybridized in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS at 65°C and washed.

[0039] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0040] In the above application, the expression cassette containing nucleic acid molecules (OSKM gene expression cassette) described in B2) refers to a DNA capable of expressing the above OSKM protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing the nucleic acid molecule of any of the above proteins. The regulatory sequence can guide the coding sequence to express any of the above proteins in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence can be operably connected to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an mRNA non-translated region that is very important for the translation of the host cell. The leader sequence can be operably connected to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the protein, which can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used can be used in the present invention. It may also be necessary to add a regulatory sequence that can regulate protein expression according to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification. In these examples, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequence.

[0041] Existing expression vectors can be used to construct a recombinant vector containing the OSKM gene expression cassette.

[0042] In the above applications, the vector may be a plasmid, a cosmid, a phage or a viral vector (such as a lentiviral vector).

[0043] B3) The recombinant vector may be specifically PLE4-OSKM. The PLE4-OSKM is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the OSKM gene shown in SEQ ID No.1 in the sequence table. PLE4-OSKM can express the OSKM protein shown in SEQ ID No.2.

[0044] In the above applications, the microorganism may be yeast, bacteria, algae or fungi.

[0045] In the above application, the osteoarthritis may be senile osteoarthritis or pathological osteoarthritis.

[0046] The treatment and / or prevention of osteoarthritis may be manifested in reducing the level of inflammation in cartilage.

[0047] The present invention also provides the use of the reprogramming four-factor OSKM or the substance that increases the content or activity of OSKM in the preparation of a product for delaying joint aging.

[0048] The joint aging may be aging of articular cartilage.

[0049] The delay of joint aging can be reflected in increasing the biological activity of cartilage tissue cells, delaying the aging of joint cartilage, improving the athletic ability of aging animals, inhibiting bone hyperplasia, promoting the increase of cartilage tissue, inhibiting synovial hyperplasia and / or increasing the cells that secrete the extracellular matrix necessary for cartilage.

[0050] The present invention also provides a product, the active ingredient of which is the reprogramming four-factor OSKM or the substance that increases the content or activity of OSKM.

[0051] The product can be used to treat and / or prevent osteoarthritis and delay joint aging.

[0052] The experiments of the present invention confirm that OSKM or substances that increase the content or activity of OSKM have a clear effect in preventing articular cartilage aging and treating osteoarthritis. The present invention provides a new strategy for developing gene therapy for preventing articular cartilage aging and treating osteoarthritis, and expands the range of options for clinical gene therapy and drug therapy.

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1To increase the content of OSKM for the treatment of osteoarthritis in aged mice. Figure (A) Schematic diagram of evaluating the therapeutic effect of OSKM on osteoarthritis in aged mice. Figure (B) Real-time fluorescence quantitative PCR (chain polymerase reaction) was used to detect the transcription levels of Oct4, Sox2, Klf4 and C-Myc in the articular cartilage of mice injected with PLE4-OSKM virus. Figure (C) After the injection of PLE4-OSKM virus, the changes in the grip strength of the hind limbs of mice were detected. Figure (D) CT scan was used to evaluate the bone volume of the hind limb joint hyperplasia of aged mice after the injection of PLE4-OSKM virus. (E) Safranin fast green staining was used to evaluate the area of ​​articular cartilage in the hind limbs of aged mice after the injection of PLE4-OSKM virus. Figure (F) H&E staining was used to evaluate the synovial hyperplasia in the hind limb joints of aged mice after the injection of PLE4-OSKM virus. Figure (G) Immunohistochemical staining was used to detect the proportion of P21-positive cells in the articular cartilage of the hind limbs of aged mice after the injection of PLE4-OSKM virus. Figure (H) Immunohistochemical staining detected the proportion of MMTV-positive cells in the articular cartilage of the hind limbs of old mice after injection of PLE4-OSKM virus. Figure (I) Immunohistochemical staining detected the proportion of IL-6-positive cells in the articular cartilage of the hind limbs of old mice after injection of PLE4-OSKM virus. Figure (J) Immunohistochemical staining detected the proportion of TNFα-positive cells in the articular cartilage of the hind limbs of old mice after injection of PLE4-OSKM virus. Figure (K) Immunohistochemical staining detected the proportion of Aggrecan-positive cells in the articular cartilage of the hind limbs of old mice after injection of PLE4-OSKM virus. Figure (L) Immunohistochemical staining detected the proportion of MMP13-positive cells in the articular cartilage of the hind limbs of old mice after injection of PLE4-OSKM virus. The scale bars in Figures EL are 40μm (upper panel) and 10μm (lower panel).

[0055] Figure 2To increase the content of OSKM for the treatment of the ACLT mouse osteoarthritis model. Figure (A) Schematic diagram for evaluating the therapeutic effect of OSKM on the ACLT mouse osteoarthritis model. Figure (B) CT scan to evaluate the bone volume of the hind limb joint hyperplasia of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (C) Safranin fast green staining to evaluate the OARSI score of the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (D) H&E staining to evaluate the synovial hyperplasia of the hind limb joints of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (E) Immunohistochemical staining to detect the proportion of Aggrecan-positive cells in the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (F) Immunohistochemical staining to detect the proportion of MMP13-positive cells in the hind limb articular cartilage of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. Figure (G) Immunohistochemical staining detected the proportion of IL-6 positive cells in the articular cartilage of the hind limbs of ACLT osteoarthritis model mice after injection of PLE4-OSKM virus. The scale bars in Figures CG are 40 μm (upper panel) and 10 μm (lower panel). DETAILED DESCRIPTION

[0056] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out in accordance with the techniques and conditions described in the literature published in the art or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The quantitative tests in the following examples were all set up with no less than 10 animals. All animal experiments were approved by the Animal Welfare Committee of the Institute of Zoology, Chinese Academy of Sciences. The experimental data are expressed as mean ± standard deviation and analyzed using GraphPad Prism 9 statistical software. P < 0.05 is considered to be significantly different between groups.

[0057] OSKM chimeric mice: described in “Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, Li M, Lam D, Kurita M, Beyret E, Araoka T, Vazquez-Ferrer E, Donoso D, Roman JL, Xu J, Rodriguez Esteban C, G, Delicado E, Campistol JM, Guillen I, Guillen P, Izpisua Belmonte JC. In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell. 2016 Dec 15; 167(7): 1719-1733.e12.doi: 10.1016 / j.cell.2016.11.052.” In the article, the public can obtain the biological material from the applicant, and the biological material can only be used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0058] Human embryonic kidney 293T (HEK) cell line: ATCC product, catalog number CRL-3216.

[0059] HEK293T cell culture medium: 89% DMEM high glucose medium (HyClone), 10% fetal bovine serum (Gibco, 10100-147), 1% penicillin / streptomycin (Invitrogen, 15070-063).

[0060] Example 1: Therapeutic effect of increasing OSKM content on osteoarthritis in aged mice

[0061] In this example, a lentiviral vector expressing OKSM was injected into the joint cavity of aged mice to delay the aging of articular cartilage and alleviate the symptoms of osteoarthritis. The experimental steps are as follows:

[0062] 1. Preparation of Lentivirus Expressing Mouse OSKM

[0063] 1.1 Preparation of recombinant vectors for increasing mouse OSKM gene

[0064] The template for amplifying the OSKM tandem expression sequence (Oct4-P2A-Sox2-T2A-Klf4-E2A-CMyc) was liver tissue DNA from OSKM chimeric mice, and the amplification primers used were as follows:

[0065] cDNA-OSKM-F: 5'-ATGGCTGGACACCTGGCTTCAGACTTCGCCTTCTC-3';

[0066] cDNA-OSKM-R: 5'-TTATGCACCAGAGTTTCGAAGCTGTTCGAGTTTGT-3'.

[0067] The full-length cDNA sequence of mouse OSKM obtained by PCR amplification (can also be directly synthesized, the sequence is SEQ ID No.1 in the sequence list, encoding the OSKM protein shown in SEQ ID No.2) is connected to the PLE4 vector double-digested with BamHI (NEB) and MluI (NEB), and the recombinant vector with the correct sequence is recorded as the recombinant vector PLE4-OSKM. PLE4-OSKM is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the OSKM gene shown in SEQ ID No.1 in the sequence list, and PLE4-OSKM can express the OSKM protein shown in SEQ ID No.2.

[0068] In SEQ ID No. 1, the 1st to 1056th positions are Oct4 gene, the 1057th to 1134th positions are P2A gene, the 1135th to 2091th positions are Sox2 gene, the 2092th to 2193th positions are T2A gene, the 2194th to 3615th positions are Klf4 gene, the 3616th to 3744th positions are E2A gene, and the 3745th to 5061th positions are C-Myc gene. In SEQ ID No. 2, the 1st to 352th positions are Oct4, the 379th to 697th positions are Sox2, the 732th to 1205th positions are Klf4, and the 1249th to 1687th positions are C-Myc.

[0069] According to the above method, a control recombinant vector (PLE4-GAL4) with correct sequence was obtained. The primers used are as follows:

[0070] cDNA-GAL4-F: 5'-ATGAAGCTACTGTCTTCTATC-3';

[0071] cDNA-GAL4-R: 5'-CTAAACCTTTCTCTTTTTCTTGG-3'.

[0072] PLE4-GAL4 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and MluI recognition sequences of the PLE4 vector with the GAL4 gene shown in SEQ ID No. 3 in the sequence list. PLE4-GAL4 can express the GAL4 protein.

[0073] 1.2 Preparation of PLE4-OSKM lentiviral concentrate

[0074] The PLE4-OSKM recombinant vector was co-transfected with the lentiviral packaging vector psPAX2 (Addgene product, #12260) and pMD2.G (Addgene product, #12259) into 293T cells to obtain the lentivirus containing PLE4-OSKM. The steps are as follows:

[0075] 1.2.1 Lipo3000 transfection kit (Thermo Scientific) was used to co-transfect 293T cells with PLE4-OSKM recombinant vector, lentiviral packaging vector psPAX2 and pMD2.G (ratio: 1 10 cm dish of 293T cells: 9 μg PLE4-OSKM recombinant vector, 6 μg psPAX2 and 3 μg pMD2.G) and incubated in a 37°C incubator.

[0076] 1.2.2 After 8 hours of culture, replace with fresh 293T cell culture medium and continue to culture for 48-54 hours.

[0077] 1.2.3 Collect the culture supernatant, filter it with a 0.45 μm pore size filter membrane, and collect the filtrate.

[0078] 1.2.4 Centrifuge at 19,400 rpm for 2 hours at 4°C, discard the supernatant, and resuspend in PBS to obtain the PLE4-OSKM-containing lentiviral concentrate, referred to as PLE4-OSKM lentiviral suspension.

[0079] According to the above method, PLE4-OSKM was replaced with PLE4-GAL4 to obtain PLE4-GAL4 lentiviral suspension, and the lentivirus was resuspended in PBS.

[0080] 2. Establishment of osteoarthritis model in naturally aged mice

[0081] Test mice: Male, 18-month-old SPF mice (C57BL / 6J), from Beijing Biotechnology Co., Ltd. Mice were kept at 23°C, 12 hours of light / 12 hours of darkness, free access to food and water. Experimental equipment and materials included: isoflurane gas anesthesia apparatus, insulin syringe.

[0082] Grouping and treatment: The mice were randomly divided into two groups, with 11 mice in each group.

[0083] In the PLE4-GAL4 group (11 mice), 20 μl of PLE4-GAL4 lentiviral suspension was injected into the left and right joint cavities of the hind limbs of each mouse.

[0084] In the PLE4-OSKM group (11 mice), 20 μl of PLE4-OSKM lentiviral suspension was injected into the left and right joint cavities of the hind limbs of each mouse.

[0085] 3. Effect evaluation

[0086] 3.1 Supplementing OSKM content delays joint aging, delays aging of joint cartilage, and improves arthritis symptoms

[0087] The lentiviral suspension was injected into the hind limb joint cavity of aged mice, and the therapeutic effect was evaluated 8 weeks later ( Figure 1 The specific method is as follows:

[0088] 3.1.1 Behavioral evaluation of changes in joint mobility of mice

[0089] Eight weeks after the injection of the lentiviral suspension, the joint mobility of the mice was evaluated by testing the grip strength of their hind limbs.

[0090] Grip strength test: Use a grip strength tester to test. Keep the mouse's trunk parallel to the grid, let its hind legs grasp the tester, grab the mouse's tail and pull it back evenly, and record the maximum grip strength value. Repeat 10 times for each mouse, and take the average value as the final grip strength value of the mouse.

[0091] The results showed that compared with mice injected with PLE4-GAL4 lentivirus suspension, mice injected with PLE4-OSKM lentivirus suspension had significantly increased grasping strength ( Figure 1 Middle C) shows that the motor ability of mice injected with PLE4-OSKM lentivirus suspension was improved.

[0092] 3.1.2 Detection of OSKM (Oct4, Sox2, Klf4 and C-Myc) expression levels in cartilage tissue

[0093] The right hind knee cartilage tissue of PLE4-GAL4 and PLE4-OSKM mice was lysed with TRIzol (Invitrogen, 15596018) and RNA was extracted. RT-qPCR was used to detect the transcription (mRNA) levels of Oct4, Sox2, Klf4 and C-Myc. The primer sequences are as follows:

[0094] Oct4-F: 5'-GGCTTCAGACTTCGCCTTCT-3';

[0095] Oct4-R: 5'-TGGAAGCTTAGCCAGGTTCG-3';

[0096] Sox2-F: 5'-TTTGTCCGAGACCGAGAAGC-3';

[0097] Sox2-R: 5'-CTCCGGGAAGCGTGTACTTA-3';

[0098] Klf4-F: 5'-GCACACCTGCGAACTCACAC-3';

[0099] Klf4-R: 5'-CCGTCCCAGTCACAGTGGTAA-3';

[0100] C-Myc-F: 5'-ACCACCAGCAGCGACTCTGA-3';

[0101] C-Myc-R: 5'-TGCCTCTTCTCCACAGACACC-3';

[0102] Compared with mice injected with PLE4-GAL4 lentivirus suspension, the mRNA levels of Oct4, Sox2, Klf4, and C-Myc in the cartilage tissue of mice injected with PLE4-OSKM lentivirus suspension were significantly increased ( Figure 1 Middle B) shows that the PLE4-OSKM lentiviral suspension was injected into the joint cavity, successfully infecting chondrocytes and increasing the expression levels of Oct4, Sox2, Klf4 and C-Myc.

[0103] 4. Effect evaluation

[0104] Eight weeks after the PLE4-GAL4 and PLE4-OSKM lentiviral suspensions were injected into the left hind knee joint of mice, the muscle tissue was removed, and the femur and tibia with a length of 1 cm were retained and fixed in 4% paraformaldehyde solution for CT detection and tissue embedding and staining. The specific steps are as follows:

[0105] 4.1 CT Scan

[0106] The knee joints were fixed in 4% paraformaldehyde and scanned using a small animal CT (Quantum GX2) at a voltage of 52 kVp, a current of 100 μA, and a resolution of 36 μm. The osteophyte volume was analyzed using Analyze (V14.0) and Slicer (V5.2) software.

[0107] CT scan data showed that compared with mice injected with PLE4-GAL4 lentivirus suspension, the bone volume (BV) of the joints of mice injected with PLE4-OSKM lentivirus suspension was significantly reduced ( Figure 1 D). This indicates that supplementing the content of OSKM in the joint cavity helps inhibit bone hyperplasia.

[0108] 4.2 Safranin Fast Green Staining of Articular Cartilage

[0109] (1) After dewaxing and rehydration, the sections were stained with fresh hematoxylin solution at room temperature for 3-5 minutes.

[0110] (2) After differentiation with 1% hydrochloric acid / ethanol for 15 seconds, wash with distilled water for 10 minutes.

[0111] (3) Place in Fast Green staining solution and stain for 5 minutes at room temperature.

[0112] (4) Quickly rinse with a weak acid solution for 10-15 seconds to remove the remaining fast green and dry.

[0113] (5) Place in safranin staining solution and stain for 5 minutes at room temperature.

[0114] (6) Dehydration, transparency, and sealing.

[0115] (7) Scan and count.

[0116] Safranin fast green staining results showed that compared with mice injected with PLE4-GAL4 lentivirus suspension, the cartilage area of ​​mice injected with PLE4-OSKM lentivirus suspension was significantly increased ( Figure 1 Middle E). This indicates that increasing the content of OSKM in cartilage tissue can help promote the increase of cartilage tissue.

[0117] 4.3 Hematoxylin-eosin (H&E) staining of articular cartilage tissue

[0118] (1) After dewaxing and rehydration, the sections were stained with fresh hematoxylin solution at room temperature for 3-5 minutes.

[0119] (2) After differentiation with 1% hydrochloric acid / ethanol for 15 seconds, wash with distilled water for 10 minutes.

[0120] (3) Place in eosin staining solution and stain for 5 minutes at room temperature.

[0121] (4) Rinse with clean water for 2 minutes.

[0122] (5) Dehydration, transparency, and sealing.

[0123] (6) Scan and count.

[0124] The results of H&E staining showed that the synovial thickness of mice injected with PLE4-OSKM lentivirus suspension was significantly reduced compared with that of mice injected with PLE4-GAL4 lentivirus suspension ( Figure 1 (F) This indicates that supplementing the content of OSKM in the joint cavity helps to inhibit synovial hyperplasia.

[0125] 4.4 Immunohistochemical staining of articular cartilage tissue

[0126] (1) Take the fixed joint tissue and decalcify it using a decalcification solution containing 5% formic acid at 4°C until the femoral and tibial tissues soften and can be punctured with a needle (approximately 7-10 days).

[0127] (2) Embed in paraffin, slice to a thickness of 5 μm, and bake at 60°C overnight.

[0128] (3) Dewax and rehydrate the sections.

[0129] (4) Use 0.125% trypsin (ZLI-9010) to repair the antigen and incubate at 37°C for 30 minutes.

[0130] (5) Permeabilize with 0.4% Triton X-100 (Sigma-Aldrich, T9284) at room temperature for 1 hour.

[0131] (6) Use an endogenous peroxidase blocker to block endogenous peroxidase.

[0132] (7) After washing with PBS at room temperature, add primary antibody and incubate at 4°C overnight.

[0133] (8) Wash with PBS at room temperature for 10 min each time, three times.

[0134] (9) Add reaction enhancement solution and incubate at room temperature for 20 minutes.

[0135] (10) Wash thoroughly with PBS at room temperature for 20 min each time, for a total of 3 times.

[0136] (11) Add enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer (Zhongshan Jinqiao Company, PV-9001 / PV-9002) and incubate at room temperature for 1 hour.

[0137] (12) Select an appropriate time for DAB color development.

[0138] (13) Hematoxylin staining, differentiation and washing, and blueing.

[0139] (14) Dehydration, transparency, and sealing.

[0140] (15) The images were scanned using a PE panoramic scanner (PerkinElmer Vectra Polaris), and Image J was used to count the proportion of positive cells in the cartilage layer.

[0141] The antibody used to detect P21 protein was produced by Cell Signaling Technology, and the product number is 2947S.

[0142] The antibody used to detect MMTV protein was produced by Novus Biologicals, with the catalog number NBP2-44179.

[0143] The antibody used to detect IL-6 protein was produced by Abcam, with the catalog number ab6672.

[0144] The antibody used to detect TNFα protein was produced by Abcam, with the catalog number ab1793.

[0145] The antibody used to detect MMP13 protein was produced by Abcam, with the catalog number ab39012.

[0146] The antibody used to detect Aggrecan protein was produced by ABclonal, with the product number A11691.

[0147] The results showed that compared with mice injected with PLE4-GAL4 lentivirus suspension, the proportion of P21 and MMTV positive cells in mice injected with PLE4-OSKM lentivirus suspension was significantly reduced ( Figure 1 G and H), IL-6 and TNFα positive cells were significantly reduced ( Figure 1 I and J), indicating that the addition of OSKM to the joint cavity significantly reduced senescent cells and inflammatory cells in the cartilage tissue. In addition, the proportion of positive cells secreting Aggrecan increased significantly ( Figure 1 K), while the proportion of positive cells secreting matrix metalloproteinase MMP13 was significantly decreased ( Figure 1 Middle (L), indicating that the amount of OSKM added to the joint cavity significantly increased the number of cells in the cartilage tissue that secrete the extracellular matrix necessary for cartilage.

[0148] Example 1 illustrates that supplementing the content of OSKM alleviates the aging of articular cartilage in aged mice, reduces the level of cartilage inflammation, and improves the biological activity of cartilage tissue cells.

[0149] Example 2: Therapeutic effect of increasing OSKM content on pathological osteoarthritis in mice

[0150] 1. Preparation of Lentivirus Expressing Mouse OSKM

[0151] Same as step 1 in Example 1.

[0152] 2. Establishment of anterior cruciate ligament injury-induced osteoarthritis (ACLT) model

[0153] 2.1 Establishment of ACLT mouse model

[0154] Test mice: female, 2-month-old SPF mice (C57BL / 6J), from Beijing Biotechnology Co., Ltd. Mice were kept at 23°C, 12 hours of light / 12 hours of darkness, free access to food and water. Experimental equipment and materials included: isoflurane gas anesthesia apparatus, insulin syringe.

[0155] The mice were randomly divided into 3 groups, with 12 mice in each group. Two groups of mice were randomly selected for ACLT modeling:

[0156] After the mice were anesthetized, the skin was cut at the knee joint of the hind limb to expose the knee joint, the anterior cruciate ligament was cut, the skin was sutured, and the ACLT mouse model was obtained after normal feeding for 10 days.

[0157] The other group did not receive any treatment and served as the control group (Sham group).

[0158] 2.2 Lentivirus injection

[0159] Sham group: no virus injection.

[0160] PLE4-GAL4 group: 20 μl of PLE4-GAL4 lentiviral suspension was injected into the left and right joint cavities of the hind limbs of each mouse.

[0161] PLE4-OSKM group: 20 μl of PLE4-OSKM lentiviral suspension was injected into the left and right joint cavities of the hind limbs of each mouse.

[0162] 3. Effect evaluation

[0163] 8 weeks after injection of lentiviral suspension ( Figure 2 A), the effect of treatment was evaluated. The specific method is as follows: the mice were killed by spinal dislocation and the hind limb knee joint was isolated. After removing the muscle tissue from the left knee joint, 1 cm of the femur and tibia were retained for CT detection and tissue embedding staining. The changes in joint tissue pathology were evaluated according to step 4 in Example 1, where the OARSI cartilage scoring criteria are as follows:

[0164] 0 = normal;

[0165] 0.5 = no structural change, a small portion of green (proteoglycan) is lost;

[0166] 1 = slight wear of the cartilage surface, with the damaged area less than 5% of the total area;

[0167] 2 = partial loss of cartilage surface matrix, not involving the deep cartilage, with the damaged area accounting for about 5%-10% of the total area;

[0168] 3 = The injury involves the calcified cartilage layer, and the damaged area accounts for about 10%-24% of the total area;

[0169] 4 = Lesion extends below the calcified cartilage, with more than 25%-50% cartilage loss on the joint surface;

[0170] 5 = 50%-75% cartilage loss on the joint surface;

[0171] 6 = Loss of greater than 75% of the cartilage on the joint surface, with possibly only a few areas of acellular collagen remaining.

[0172] CT scan data showed that the volume of osteophyte hyperplasia in the joints of mice injected with PLE4-OSKM lentivirus suspension was significantly reduced compared with mice injected with PLE4-GAL4 lentivirus suspension ( Figure 2 (B) This indicates that supplementing the content of OSKM in the joint cavity helps inhibit bone hyperplasia.

[0173] Safranin fast green staining results showed that compared with mice injected with PLE4-GAL4 lentivirus suspension, the cartilage OARSI score of mice injected with PLE4-OSKM lentivirus suspension was reduced ( Figure 2 (C) This indicates that increasing the content of OSKM in cartilage tissue can help promote the increase of cartilage tissue.

[0174] The results of H&E staining showed that the synovial thickness of mice injected with PLE4-OSKM lentivirus suspension was significantly reduced compared with that of mice injected with PLE4-GAL4 lentivirus suspension ( Figure 2 (D) This indicates that supplementing the content of OSKM in the joint cavity helps to inhibit synovial hyperplasia.

[0175] At the same time, it was found that the proportion of cells secreting Aggrecan increased significantly ( Figure 2 E), while the proportion of cells secreting matrix metalloproteinase MMP13 was significantly decreased ( Figure 2 F), indicating that the amount of OSKM added to the joint cavity increased the number of cells in the cartilage tissue that secrete the extracellular matrix necessary for cartilage. In addition, the number of IL-6 positive cells decreased significantly ( Figure 2 Middle G) indicates that the amount of OSKM added to the joint cavity reduces the inflammatory cells in the cartilage tissue.

[0176] Example 2 illustrates that supplementing the content of OSKM into the joint cavity alleviates cartilage wear caused by pathological osteoarthritis, reduces the level of joint inflammation, and improves the biological activity of cartilage tissue cells.

[0177] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of substances that reprogram four-factor OSKM or increase OSKM content or activity in the preparation of products for the treatment and / or prevention of osteoarthritis.

2. The use according to claim 1, characterized in that: OSKM is composed of or connected by Oct4, Sox2, Klf4 and C-Myc.

3. The use according to claim 1 or 2, characterized in that: OSKM is as follows A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 2; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

4. The use according to any one of claims 1 to 3, characterized in that: The substance that increases the content or activity of OSKM is a substance that promotes the expression of OSKM gene or a biological material related to OSKM; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding OSKM; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

5. The use according to claim 4, characterized in that: B1) The nucleic acid molecule is the following b11) or b12) or b13) or b14): b11) the coding sequence is the DNA molecule of SEQ ID No.1 in the sequence list; b12) the DNA molecule shown in SEQ ID No. 1 in the sequence list; b13) a DNA molecule encoding OSKM that has 75% or more identity with the nucleotide sequence defined in b11) or b12); b14) A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b11) or b12) or b13) and encodes OSKM.

6. The use according to any one of claims 1 to 5, characterized in that: The osteoarthritis is senile osteoarthritis or pathological osteoarthritis; And / or, the treatment and / or prevention of osteoarthritis is manifested in reducing the level of inflammation of cartilage.

7. Use of the reprogramming four-factor OSKM or the substance that increases the content or activity of OSKM according to any one of claims 1 to 5 in the preparation of products for delaying joint aging.

8. The use according to claim 7, characterized in that: The method of delaying joint aging is embodied in increasing the biological activity of cartilage tissue cells, delaying the aging of joint cartilage, improving the athletic ability of aging animals, inhibiting bone hyperplasia, promoting the increase of cartilage tissue, inhibiting synovial hyperplasia and / or increasing the cells that secrete the extracellular matrix necessary for cartilage.

9. A product, the active ingredient of which is the reprogramming four-factor OSKM or the substance that increases the content or activity of OSKM as claimed in any one of claims 1 to 5.

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